We investigate the molecular mechanisms by which transmembrane proteins are sorted to intracellular compartments such as endosomes, lysosomes, and a group of cell-type-specific organelles known as lysosome-related organelles, which includes melanosomes and platelet dense bodies. Sorting to these compartments is mediated by recognition of signals in the cytosolic domains of the transmembrane proteins by adaptor proteins that are components of protein coats. Among these adaptor proteins are the heterotetrameric AP1, AP2, AP3 and AP4 complexes, the monomeric GGA1, GGA2 and GGA3 proteins (i.e., the GGAs) and the heteropentameric retromer complex. Current work is aimed at elucidating the structure, regulation and physiological roles of recognition proteins, as well as investigating human diseases that result from genetic defects in these proteins (i.e., the Hermansky-Pudlak syndrome) or from their exploitation by pathogens (i.e., HIV-1).[unreadable] [unreadable] The AP complexes and GGAs have ear domains that bind accessory proteins. We previously demonstrated that these interactions are mediated by a canonical sequence motif shared by the accessory proteins. The physiological relevance of these interactions, however, had not been established. This past year, we have examined the significance of interactions of the ear domains of the AP and GGA proteins with two accessory proteins, GAK and p56. We found that depletion of both GAK and p56 by RNA interference (RNAi) resulted in missorting of mannose 6-phosphate receptors (MPRs) and their cargo lysosomal hydrolases, resulting in a phenotype similar to that of certain lysosomal storage disorders. This defect could be reversed by transfection with the wild-type GAK or p56, but not with mutant GAK or p56 with substitution of their ear-binding motifs. This demonstrated for the first time that the ability of AP complexes and GGAs to engage in canonical ear-motif interactions is critical to their function in protein sorting and lysosome biogenesis.[unreadable] [unreadable] Newly-made acid hydrolases are sorted by binding to MPRs at the TGN. The hydrolase-receptor complexes are recognized by the GGA proteins, which mediate packaging into transport vesicles bound for endosomes. The acidic environment of endosomes induces the release of the hydrolases from the MPRs, after which the hydrolases follow the fluid phase to lysosomes, while the MPRs return to the TGN to mediate further rounds of transport. In previous work, we showed that the proteins, Vps26, Vps29 and Vps35, which are subunits of a protein complex named retromer, played a role in this retrograde transport of MPRs from endosomes to the TGN. We have recently examined the requirement of two other putative subunits of retromer, the sorting nexins 1 and 2 (SNX1 and SNX2). Using RNA interference, we found that depletion of either SNX protein by RNAi had no effect on MPR trafficking, but combined depletion of both SNX proteins impaired the recycling of MPRs to the TGN and caused its missorting to lysosomes, where they were degraded. As a consequence, lysosomal enzymes were missorted into the extracellular space, a phenotype that is typical of lysosomal storage disorders. These findings demonstrated that SNX1 and SNX2 play interchangeable but essential roles, as part of the retromer complex, in the sorting of MPRs from endosomes to the TGN.[unreadable] [unreadable] To elucidate the structural bases for the role of retromer in MPR retrograde transport, we collaborated with James Hurley (NIDDK), Alasdair Steven (NIAMS), and their co-workers, to solve the structure of the retromer Vps26-Vps29-Vps35 subcomplex by X-ray crystallography and electron microscopy. We found that this complex is a 21nm-long rod having Vps26 at one end and Vps29 at the other. Vps26 is structurally similar to arrestins, whereas Vps29 resembles a type of metallophophoesterase. Vps35 consists of a long alpha-helical solenoid that spans the length of the rod and covers the putative metallophosphoesterase active site on Vps29. Vps35 also exposes multiple grooves that could be binding sites for sorting signals on cargo molecules such as the MPRs.[unreadable] [unreadable] The retromer complex is not only used to retrieve intracellular sorting receptors from endosomes to the TGN, but is also exploited by certain bacterial toxins to access their target compartments. An example is Shiga toxin, which we have shown, in collaboration with Ludger Johannes and Graa Raposo (Curie Institute, Paris), to require retromer for its movement from endosomes to the TGN. This transport begins at vacuolar early endosomes and proceeds through tubules that are part of what we refer as the tubular endosomal network.[unreadable] [unreadable] The sorting of integral membrane proteins into the lumen of lysosomes involves passage through an intermediate organelle known as the multivesicular body (MVB). For most proteins, this sorting involves recognition of ubiquitinated proteins by several complexes known as ESCRT. In collaboration with James Hurley (NIDDK) and colleagues, we have solved the crystal structure of the ESCRT Vps27-Hse1 complex. The core of this complex is constituted by two intertwined GAT domains, each consisting of two helices from one subunit and one from the other. These domains are similar to the previously described GAT domain of the GGAs. The two Vps27-Hse1 GAT domains are connected by an antiparallel coiled-coil to form a 90 -long barbell-like structure. These studies explain how the complex binds cooperatively to lipids and ubiquitinated membrane proteins and acts as a scaffold for ubiquitination reactions.[unreadable] [unreadable] Although most proteins require ubiquitination for targeting to the MVB pathway, the yeast transmembrane protein Sna3p is an exception. Despite the fact that Sna3p itself is not ubiquitinated, we have found that the ubiquitin ligase Rsp5 and the ESCRT complexes are nonetheless required for Sna3p targeting to the MVB pathway. This targeting is mediated by a direct interaction between a PPAY motif within the Sna3p C-terminal cytosolic domain and the WW domains of Rsp5p. Sna3p is thus an example of a new class of proteins that follow a ubiquitination-independent, but ubiquitin-ligase-mediated, sorting pathway to the vacuole.